The recent employment of matrix-assisted-laser-desorption/ionization (MALDI) (Karas & Hillenkamp, Anal. Chem. 60, 2299 (1988)) with time-of-flight (TOF) mass spectrometry has extended the accuracy of mass spectrometric measurements to include proteins and nucleic acids. See generally, Kinter, Anal. Chem. 67, 493R-497R (1995); Schoneich et al, Anal. Chem. 67, 155R-181R (1995); Busch, J. Chromatog. A 692, 275-290 (1995); and Limbach et al, Curr. Opin. Biotechnol. 6, 96-102 (1995). Today, the sensitivity, mass range and ability to analyze complex mixtures has made mass spectrometry an important tool for the analysis of large biomolecules. Many researchers both in academic and industrial settings use MALDI as an integral part of biomolecule analyzing experimentation.
MALDI has many advantages over traditionally employed methods of large biomolecule analyzation, such as two-dimensional gel electrophoresis. For example, MALDI displays both high sensitivity and relatively high tolerance to the presence of sample contaminants. MALDI also has the ability to accurately measure biomolecules using only subpicomole amounts of analyte. Furthermore, MALDI may be automated, thus, making the technique less labor intensive and time consuming as compared to traditional analyzation methods. MALDI automation also allows handling of small analyte volumes, an advantage sought by many MALDI users. Furthermore, MALDI provides a means for repeat analyzation of a particular sample as it is destructive to only a minute portion of the sample. Thus, it leaves a massive amount of sample available for re-analysis. Other current techniques such as electrospray are sample destructive leaving no sample for repeat analysis.
Although MALDI has currently been automated to a certain degree, the completely successful automation of MALDI spotting, because of the extensive and exacting analyte sample preparation required, has proven difficult. Generally, in order to correctly prepare a MALDI analyte sample, MALDI matrix solution must be added to the analyte prior to analysis. Even following this addition, interpretable results necessitate the MALDI matrix solution and analyte be evenly distributed to produce a homogeneous analyte sample. Consequently, complete mixing of MALDI matrix solution and analyte is required to obtain a reliable analyte sample. As used herein, an analyte sample is a combination of analyte and an appropriately absorbing sample matrix solution.
In automated MALDI spotting, because the analyte sample is not vortexed, it is often difficult to achieve the degree of homogenization that results from the complete mixing of MALDI matrix solution and analyte during manual spotting. Complete homogenization is important as complete mixing provides a higher quality spot and eliminates “hot spots” in the target, making the MALDI target more uniform. The elimination of hot spots is essential to accurate MALDI experimentation, regardless of whether manual or automated MALDI is being performed. Although these hot spots may be eliminated by thorough vortexing in manual MALDI, a need exists to insure complete mixing of analyte sample when using automated MALDI spotting. In automated MALDI spotting, an important step toward ensuring complete mixing includes incorporating the use of mixing chambers. Using a mixing chamber during automated MALDI spotting increases the mixing of analyte and matrix solution, resulting in increased signal to noise ratios and fewer “hot spots.”
Unfortunately, in the past, when these mixing chambers were used with particular types of sample dispensing probes, such as MALDI target spotting probes, certain problems arose. For example, when a mixing chamber is used with a spring loaded probe, because the tubing in the spring loaded probe moves during spotting of the MALDI plate, the tubing may easily kink and bend. This distortion of the tubing may result in MALDI targets that are not uniform, making the results harder to interpret. Furthermore, because of the extra weight placed on the tubing when a mixing chamber is used without a mixing chamber probe adaptor, the tubing may separate from the mixing chamber, either resulting in lost analyte, MALDI matrix, or in the case of tubing that separates the mixing chamber from the probe, lost analyte sample. But because spring loaded probes provide many advantages, such as eliminating direct contact between the probe and the MALDI plate and allowing accurate MALDI target deposition by handling small amounts of analyte sample with minimal loss, it is preferable to use spring loaded probes over other sample dispensing probes during automated MALDI spotting. The spring in the spring loaded probe allows tubing, not the spring loaded probe itself, to touch the MALDI plate when spotting a MALDI target. Although glass capillaries that directly touch the plate may be used to spot MALDI targets, their fragility and propensity to cause MALDI plate damage makes them less desirable than a spring loaded probe. Spring loaded probes cause less damage to both the probe itself and the MALDI plate. The use of spring loaded tubing to spot a MALDI target also results in more accurate MALDI targets, as a greater amount of a more uniform pattern of analyte sample is added to the MALDI plate. In addition, the spring loaded probe permits a lower dead volume in the system as a result of the tubing in the spring loaded probe directly touching the MALDI plate, thus eliminating analyte sample loss caused by probe surface tension. Consequently, a need exists for a solution to the problems created when using a mixing chamber with a spring loaded probe.